Project Summary Influenza pandemics are caused by emergent novel influenza A viruses (IAVs) that transmit efficiently within human populations lacking preexisting immunity against the specific virus. After the significantly divergent genome sequences of novel HL17NL10 and HL18NL11 bat influenza A-like viruses (BIALVs) were identified, concerns have been raised that they may pose significant spillover threats to humans because antibodies to IAVs and influenza B viruses have no cross-reactivity to novel BIALVs. To understand these novel viruses, reverse genetics was established for both viruses. It was demonstrated that internal genes of these viruses are functional through generation of chimeric bat viruses that contain six internal genes from the bat virus and the ORF of both HA and NA from canonical IAVs. It was also shown that reassortant viruses that carry the classical IAV M gene by replacing the bat M gene in the genetic background of chimeric bat virus can be rescued. Bats have been shown to be seropositive to IAVs, frequently to the H9N2 viruses. Furthermore, bat cells from different species have been demonstrated to support human, swine and avian IAV replication. The bats could have been exposed to both BIALVs and IAVs, and reassortment might occur to generate novel viruses that can infect other species including humans. Recent studies showed that rescued BIALVs can infect canine and human cells. All facts suggest a zoonotic potential of novel bat viruses. However, little is known about the receptors of these novel viruses, infection and immunological responses in their natural hosts (bats), or how they are maintained and transmitted among their natural hosts. Whether bats can be infected by IAVs, and if they are infected, what role in the ecology of IAVs do these infections play? Significant knowledge is needed to understand these novel viruses and their potential threats to other species including humans. Jamaican fruit bats (Artibeus jamaicensis) were shown to be experimentally susceptible to the rescued wild type HL18NL11 virus. Therefore, it is hypothesized that Jamaican fruit bats can be used as a model organism for understanding novel BIALVs and their potential threats to other species including humans. This proposal includes three specific aims: 1) To determine BIALV infection kinetics and tropisms in bats, as well as identify cellular receptors; 2) To determine reassortment potential and mechanisms between BIALVs and classical IAVs; 3) To determine which bat adaptive immune responses are critical to controlling BIALV infection. The results from this proposal will provide novel insights into the biology and virology of novel BIALVs, reveal the association of identified viral sequences with bats, identify roles that bats may play in virus ecology, and address concerns regarding their potential threats to other species including humans, which are important for the both influenza and bat immunity research communities.